Rhabdomyosarcomas are highly malignant tumors composed of primitive muscle cells (stem cells) having a low propensity to differentiate. Cells of this type are grouped by histologic and cytogenetic criteria as either embryonal or alveolar rhabdomyosarcomas. The two types are distinguished by detection in embryonal rhabdomyosarcomas of a loss of heterozygosity on the short arm of chromosome 11 encompassing 11p15.5 [Mitchell et al, Oncogene, 6:89-92 (1991)] and in alveolar rhabdomyosarcomas, of a balanced translocation between chromosomes 2 and 13, t(2:13)(q35;q14) [Barr et al., Nat. Genet., 3:113-117 (1993)]. Loss of heterozygosity at 11p15.5 is also associated with a number of other solid tumors [Newsham et al., Genes Chromosom. Cancer, 3:108-116 (1991)] suggesting the location of a tumor suppressor gene(s) for multiple tumor types in this region.
In tumor cells, expression of the muscle differentiation factor MyoD [Weintraub, H., Cell, 75:1241-1244 (1993)] has been shown to be a highly sensitive marker for classifying sarcomas as rhabdomyosarcomas [Dias et al., Am. J. Pathol., 137:1283-1291 (1990); Scrable et al., Proc. Natl. Acad. Sci., USA, 87:2182-2186 (1990)]. MyoD is a member of a large family of transcription factors that belong to the basic-helix-loop-helix (BHLH) family known to control cell fate determination and stem cell function. While MyoD is associated with myoblast differentiation, related proteins determine the fate of other primitive cell types. For example, SCL controls Hematopoietic stem cell differentiation [Porcher, et al., Cell 86:47-57 (1996)] and neurogenic stem cell differentiation is controlled by the BHLH proteins MASH, neurogenin, and neuro D [Reviewed in Morrison et al., Cell 88:287-298 (1997) and Andersen, FASEB J., 8:707-713 (1994)]. Similarly, liver stem cell differentiation is regulated by a combination of transcription factors including NF-.kappa.B, Stat3, and C/EBP [Taub, FASEB J. 10:413-427 (1996)]. Conversely, expression of transforming oncogenes inhibits cellular differentiation in several different cell lineages [Holtzer et al., Proc. Natl. Acad. Sci. USA, 72:4051-4055 (1975); Lassar et al., Cell, 58:659-667 (1989)]. In muscle cells, for example, expression of oncogenic tyrosine kinases (v-src and v-fps), growth factor receptors (v-erbB), nuclear oncogenes (v-myc, c-myc, v-erbA, E1A, and MDM2), and the activated form of signal transducing G proteins (H-ras and N-ras) can inhibit terminal differentiation to varying degrees [Fiszman and Fuchs, Nature, 254:429-431 (1975); Holtzer et al., Proc. Natl. Acad. Sci. USA, 72:4051-4055 (1975); Fiddler et al., Mol. Cell Biol. 16:5048-5057 (1996)]. The paradox that MyoD, shown to induce muscle differentiation in a wide variety of primary cells and transformed cell lines [Weintraub et al., Proc. Natl. Acad Sci., 86:5434-5438 (1989)], serves as a hallmark for identification of a particular tumor type may be resolved by the possibility that MyoD appears to be non-functional in the neoplastic cells.
In rhabdomyosarcomas, abnormalities in protein expression have been reported, including for example, p53 and ras expression [Hiti et al., Mol. Cell Biol., 9:4722-4730 (1989); Dias et al., Am. J. Pathol., 137:1283-1291 (1990), Loh et al. Proc. Natl. Acad Sci. USA, 89:1755-1759 (1992)], however, the loci involved in the 11p loss of heterozygosity have not been identified. It is clear, however, that MyoD expression is unaffected [Scrable et al, Proc. Natl. Acad Sci., USA, 87:2182-2186 (1990)]. Chromosome transfer experiments wherein a normal chromosome 11 was introduced into rhabdomyosarcoma cells resulted in inhibition of cell growth and tumor formation in nude mice but had no effect on myogenic differentiation [Loh et al., Proc. Natl. Acad. Sci. USA, 89:1755-1759 (1992)]. Thus, the loss of the chromosome 11 locus was shown not to be responsible for the lack of differentiation in embryonal rhabdomyosarcomas.
One obvious phenotype of rhabdomyosarcomas is a lack of terminal differentiation and somatic cell genetic experiments have been carried out in attempts to identify genetic loci present in rhabdomyosarcoma cells that inhibit muscle differentiation. Results indicated that rhabdomyosarcomas could be classified by cell fusion experiments as either recessive or dominant with respect to their inability to differentiate [Tapscott et al., Science, 259:1450-1453 (1993)]. Furthermore, transfer of a derivative chromosome 14 from the rhabdomyosarcoma cell line Rh18, a cell type that displays a dominant non-differentiating phenotype, into the differentiation competent myoblast cell line C2C12 inhibited muscle differentiation as well as the ability of MyoD to transactivate reporter constructs. The derivative chromosome 14 contained amplified DNA originating from chromosome 12q13-14, a region containing several genes often amplified in sarcomas. Testing the amplified genes for the ability to inhibit muscle-specific gene expression indicated that forced expression of one gene in particular, Murine Double Minute Gene 2 (MDM2), inhibited MyoD function and consequently inhibited muscle differentiation [Fiddler et al., Mol. Cell Biol. 16:5048-5057 (1996)]. MDM2 was originally identified in a spontaneously transformed cell line [Fakharzadeh et al., EMBO J., 10:1565-1569 (1991)] and was subsequently shown to interact with p53 [Oliner et al., Nature, 358:80 (1992)].
In addition to the above chromosomal abnormalities, chromosome 3q alterations have been found to occur frequently in rhabdomyosarcomas. Previous studies have shown that gain of 3q was present in two out often embryonal rhabdomyosarcomas [Weber-Hall et al., Cancer Res., 56:3220-3224 (1996)]. In addition, gain of 3q has been observed at high frequency in several other types of tumors, including, for example, 52% of prostate tumors [Cher et al., Cancer Res., 56:3091-3102 (1996)], ten out of thirteen small cell lung carcinomas [Ried et al., Cancer Res., 54:1801-1806 (1994)], ten out of thirteen head and neck squamous cell carcinomas [Speicher et al., Cancer Res., 55: 1010-1013 (1995)], and nine out of ten cervical carcinomas [Heselmeyer et al., Proc. Natl. Acad. Sci. USA, 93:479-484 (1996)]. Furthermore, the gain of chromosome 3q by isochromosome formation in HPV16-infected cells defines the transition from severe dysplasia to invasive carcinoma of the uterine cervix [Heselmeyer et al., Proc. Natl. Acad. Sci. USA, 93:479-484 (1996)].
Even though the precise consequences of the various rhabdomyosarcoma genetic changes are unclear, the chromosomal rearrangement/damage is consistent with the belief that tumorigenesis is a multistep process with genetic damage occurring in most, if not all, cancer cells [Bishop, J. M., Science, 235:305-311 (1987)]. Whatever the precise mechanism involved, the prevalence of DNA damage in numerous cancer cells brings into question the role of cell cycle checkpoints in neoplastic cell types. Cell cycle checkpoints consist of signal transduction cascades which couple DNA damage detection to cell cycle progression and failure of one or more components in the system predisposes an individual to, or directly causes, many disease states such as cancer, ataxia telangiectasia, embryo abnormalities, and various immunological defects associated with aberrant B and T cell development. Polypeptides of the checkpoint system play roles in detecting and signaling a response to DNA damage that occurs as a result of replication errors, DNA mismatches, radiation damage, or chemotherapy.
It has been proposed that cell cycle checkpoints comprise at least three distinct classes of polypeptides which act sequentially in response to cell cycle signals or defects in chromosomal mechanisms. [Carr, A. M., Science, 271:314-315 (1996)]. The first class of proteins, exemplified by ATM [Rotman and Shiloh, Cancer Surv. 29:285-304 (1997)] and ATR [Keegan et al., Genes and Devel., 10:2423-2437 (1996)], detect or sense DNA damage or abnormalities. The second class of polypeptides, exemplified by Rad53. [Flaggs et al., Current Biology, (1997)], amplifies and transmits signals from the detector polypeptides. Finally, effector polypeptides, exemplified by mammalian p53, yeast weel, S. pombe CHK1, [Al-Khodairy et al., Mol. Biol. Cell, 5:147-160 (1994)], and human CHK1, bring about an appropriate cellular response, e.g. arrest of mitosis/meiosis or apoptosis.
Despite the roles for the checkpoint system to properly manage DNA damage and/or abnormalities, it is unclear what, if any relationship exists between the checkpoint system and neoplastic growth that results, at least in part, from genetic damage and/or rearrangement. More importantly, the art is silent with respect to what, if any relationship exists between expression of MyoD and the checkpoint system.
There thus is a need in the art for promoting differentiation of differentiation-inhibited cells such as rhabdomyosarcomas. More generally, there is also a need to modulate differentiation of other cells of interest. For example, inhibition of the differentiation of stem cells is contemplated so that the stem cell population may be expanded for therapeutic manipulation.